Biosensor utilizing a resonator having a functionalized surface

ABSTRACT

Systems and methods for detecting the presence of biomolecules in a sample using biosensors that incorporate resonators which have functionalized surfaces for reacting with target biomolecules. In one embodiment, a device includes a piezoelectric resonator having a functionalized surface configured to react with target molecules, thereby changing the mass and/or charge of the resonator which consequently changes the frequency response of the resonator. The resonator&#39;s frequency response after exposure to a sample is compared to a reference, such as the frequency response before exposure to the sample, a stored baseline frequency response or a control resonator&#39;s frequency response.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 10/749,529filed Dec. 30, 2003, now abandoned. The disclosure of the priorapplication is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to biosensors, and more particularly tobiosensors that incorporate resonators having functionalized surfacesfor binding or otherwise reacting with target biomolecules in a mannerthat changes the frequency responses of the resonators.

2. Background Information

Biosensors are used to detect the presence and/or levels ofbiomolecules, typically in a fluid sample. For instance, biosensors maybe used to determine the levels of particular chemicals in biologicalfluids, such as blood. Specific sensors can therefore be used todetermine the levels of glucose, potassium, calcium, carbon dioxide, andother substances in blood samples.

Biosensors such as these often use an electrochemical system to detect aparticular substance of interest. The electrochemical system includessubstances such as enzymes and redox mediators to react with thesubstance of interest (the target substance) and to thereby produce ionsthat can carry a current. A set of electrodes are used to generate anelectrical potential that attracts the ions to the electrodes, creatinga circuit that can be used to measure the resulting current.

In one type of system, a biosensor includes an enzyme which isimmobilized by a membrane. The target substance in a fluid samplemigrates through the membrane and reacts with the enzyme. This formsions within the fluid sample. These ions then migrate through the fluidsample to the system's electrodes. The migration of the ions to theelectrodes generates an electrical current that is measured. Because thecurrent depends upon the concentration of the target substance in thesample, the measured current is then translated to a concentration ofthe target substance.

There are a number of problems with these conventional biosensors. Forexample, they are relatively slow. This is, at least in part, a resultof the fact that it is necessary in electrochemical biosensors to allowa certain amount of time to pass before the current resulting from theionization of the target substance in the sample is established. Onlyafter this current is allowed to establish itself can it be measured toprovide a reasonably accurate estimate of the concentration of thetarget substance.

Even after the current resulting from the ionization of the targetsubstance is established and measured, the resulting estimation of thetarget substance concentration typically is not as accurate as would bedesirable. This is a result, at least in part, of the fact that thesample being tested typically contains various other substances, some ofwhich may interfere in the process. For instance, some of these othersubstances may ionize in the sample and thereby increase the measuredcurrent, leading to an overestimation of the target substanceconcentration. Alternatively, some chemicals may react with the ions ofthe target substance, thereby reducing the measured current and causingan underestimation of the target substance concentration.

It would therefore be desirable to provide systems and methods thatenable the testing of samples to determine the presence of targetsubstances more quickly and more accurately than is typically possibleusing prior art systems and methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention may become apparent uponreading the following detailed description and upon reference to theaccompanying drawings.

FIG. 1 is a diagram illustrating the structure of an exemplary resonatorin accordance with one embodiment.

FIG. 2 is a functional block diagram illustrating a biosensor system inaccordance with one embodiment.

FIGS. 3A-3C are a set of diagrams illustrating the binding of targetmolecules to the functionalized surface of a biosensor in accordancewith one embodiment.

FIG. 4 is a flow diagram illustrating a method for detecting thepresence of target molecules in a sample in accordance with oneembodiment.

FIG. 5 is a flow diagram illustrating a method for detecting thepresence of target molecules in a sample in accordance with analternative embodiment.

FIG. 6 is a diagram illustrating the structure of an exemplary resonatorin accordance with an embodiment.

FIG. 7 is a diagram illustrating the structure of an exemplary resonatorin accordance with an embodiment.

While the invention is subject to various modifications and alternativeforms, specific embodiments thereof are shown by way of example in thedrawings and the accompanying detailed description. It should beunderstood, however, that the drawings and detailed description are notintended to limit the invention to the particular embodiments which aredescribed. This disclosure is instead intended to cover allmodifications, equivalents and alternatives falling within the scope ofthe present invention as defined by the appended claims.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

One or more of the problems outlined above may be solved by the variousembodiments of the invention. Broadly speaking, the invention comprisessystems and methods for detecting the presence of molecules (e.g.,biomolecules) in a sample using sensors that incorporate resonatorswhich have functionalized surfaces for binding or otherwise reactingwith target molecules in a manner that changes the frequency responsesof the resonators.

In one embodiment of the invention, a device includes a resonator, wherethe resonator has at least one functionalized surface which isconfigured to react with target molecules. The reaction of the targetmolecules with the functionalized surface causes changes in the massand/or charge of the resonator, stress/strain, surface energy/tensionand the like, which cause changes in the vibrational characteristics ofthe resonator. Changes in the vibrational characteristics of theresonator may be manifested through corresponding changes in electricalcharacteristics of the resonator.

In one embodiment, the resonator consists of a layer of piezoelectricmaterial and a pair of electrodes that are coupled to opposite sides ofthe layer of piezoelectric material. One of the electrodes forms thefunctionalized surface of the resonator. When an excitation signal isapplied across the electrodes, the frequency response of the resonatorcan be determined. When target biomolecules come into contact with thefunctionalized surface, the target biomolecules react (e.g., bind) withthe functionalized surface and cause changes in the mass and/orelectrostatic charge of the resonator. By determining the frequencyresponses of the resonator before and after exposure to a sample thatmay contain target biomolecules, changes in the frequency responsecorrelated to the changed mass and/or electrostatic charge can bedetermined, indicating the detection of the target biomolecules.

In one embodiment, a pair of resonators is used. Each of the resonatorsis essentially as described above, except that one of the resonators hasa functionalized surface and the other does not. The resonator that doesnot have a functionalized surface is used as a control against which theother resonator can be compared. Thus, when both resonators are exposedto a sample, any target biomolecules will affect the frequency responseof the functionalized-surface resonator, but not the resonator withoutthe functionalized surface. Any non-target molecules will equally affectboth resonators and the corresponding frequency responses, so acomparison of the two resonators will effectively cancel out any effectsresulting from non-target molecules.

In one embodiment of the invention, a method includes the steps ofproviding a resonator having a surface functionalized with a type ofbiomolecules, where the presence of target molecules causes thebiomolecules of the functionalized surface to change the frequencyresponse of the resonator, exposing the functionalized surface of theresonator to a test fluid, determining a frequency response of theresonator after the functionalized surface has been exposed to the testfluid, and determining whether the test fluid contains target moleculesbased upon the frequency response of the resonator.

In one embodiment, the method includes the additional steps of providinga second resonator that does not have a functionalized surface, exposingthe second resonator to the test fluid, determining a frequency responseof the second resonator after the second resonator has been exposed tothe test fluid, and comparing the frequency response of the secondresonator to the frequency response of the first (functionalized)resonator to determine the effect of target molecules on the frequencyresponse of the first resonator.

Numerous additional embodiments are also possible.

One or more embodiments of the invention are described below. It shouldbe noted that these and any other embodiments described below areexemplary and are intended to be illustrative of the invention ratherthan limiting.

As described herein, various embodiments of the invention comprisesystems and methods for detecting the presence of molecules in a sampleusing sensors that incorporate resonators which have functionalizedsurfaces for binding or otherwise reacting with target molecules in amanner that changes the frequency responses of the resonators.

In one embodiment, a biosensor includes a piezoelectric resonator thathas a surface which is functionalized to react with target biomolecules.The resonator consists in one embodiment of a layer of piezoelectricmaterial that has a pair of electrodes that are coupled to oppositesides of the layer of piezoelectric material. One of the electrodesforms the functionalized surface of the resonator. When an excitationsignal is applied across the electrodes, the frequency response of theresonator can be determined. When target biomolecules come into contactwith the functionalized surface, the target biomolecules react (e.g.,bind) with the functionalized surface and cause changes in the massand/or electrostatic charge of the resonator. By determining thefrequency responses of the resonator before and after exposure to asample that may contain target biomolecules, changes in the frequencyresponse correlated to the changed mass and/or electrostatic charge canbe determined, indicating the detection of the target biomolecules.

In one embodiment, a pair of resonators is used. Each of the resonatorsis essentially as described above, except that one of the resonators hasa functionalized surface and the other does not. The resonator that doesnot have a functionalized surface is used as a control against which theother resonator can be compared. Thus, when both resonators are exposedto a sample, any target biomolecules will affect the frequency responseof the functionalized-surface resonator, but not the resonator withoutthe functionalized surface. Any non-target molecules will equally affectboth resonators and the corresponding frequency responses, so acomparison of the two resonators will effectively cancel out any effectsresulting from non-target molecules.

Referring to FIG. 1, a diagram illustrating the structure of anexemplary resonator in accordance with one embodiment is shown. Theresonator illustrated in this figure comprises a film bulk acousticresonator (FBAR) device. Device 100 includes a layer of piezoelectricmaterial 110 sandwiched between electrodes 121 and 122 to form aresonator component. This resonator component is positioned with itsedges on a silicon/silicon dioxide substrate 130 (a layer of silicondioxide 131 deposited on a layer of silicon 132) in order to allow theresonator component to vibrate. The exposed surface of electrode 121 isfunctionalized with a layer 140 of biologically active or derivatizedmaterial. The biologically active or derivatized material interacts withthe target biomolecules by, for example, binding the biomolecules tolayer 140 and thereby changing the mass or electrostatic charge of thislayer and, consequently, the resonator component.

The FBAR resonator may be constructed using techniques that are known topersons of skill in the art. For example, in one embodiment, a FBARresonator may be constructed according to the following process.

First, a substrate is provided. In one embodiment, a silicon wafer isused as the substrate, although other substrate materials used insemiconductor processing (e.g., gallium arsenide) can also be used. Alayer of sacrificial material is then deposited on the substrate. Thesacrificial layer may consist of a variety of materials, such as Al, Cu,NiFe, ZnO, or other suitable materials that are known in the art. Thesacrificial layer may be deposited using any suitable process, such assputtering or vapor deposition.

A photoresist layer is then formed on top of the sacrificial layer. Apattern is then formed in the photoresist using conventional methods.The patterned photoresist forms a mask which is used to selectively etchthe sacrificial layer. More specifically, the photoresist mask covers anarea of the sacrificial layer that will later form an air gap beneaththe piezoelectric resonator component. After the sacrificial layer isetched, an insulator layer is deposited on the substrate, effectivelyreplacing the sacrificial layer that was previously etched away. Theinsulator layer can be deposited or otherwise formed using conventionalmeans.

After the insulator layer is deposited, the photoresist is removedusing, for example, a lift-off process. The portion of the insulatorlayer that is on top of the photoresist is also removed. This results ina patterned layer of insulator and sacrificial materials on top of thesubstrate. In other words, the sacrificial material is inset within theinsulator material (or vice versa) to form a pattern within this layer.This layer will form the supporting structure for the resonatorcomponent after the sacrificial material is removed in a later step.

A membrane layer may optionally be formed on top of the patternedinsulator/sacrificial layer. References below to formation of structureson top of the layer of insulator/sacrificial material should beconstrued as formation of the structures on the membrane layer if themembrane layer is used.

A conductive layer is then formed on the layer of insulator/sacrificialmaterial. This conductive layer may consist of any conductive materialsuch as a metal. Suitable metals may include Al, Au, W, Pt or Mo. Thisconductive layer is patterned to form a lower electrode of the resonatorcomponent. A layer of piezoelectric material, such as AlN or ZnO, isthen formed on top of the conductive layer. This piezoelectric layer ispatterned to form the body of the piezoelectric resonator. A secondconductive layer is then formed on top of the piezoelectric layer. Thisconductive layer is patterned to form the upper electrode of theresonator component. After the resonator component is formed in thismanner, the sacrificial material on the substrate below the resonatorcomponent is removed using, for example, a wet etch process (it may benecessary to form a via to the sacrificial layer in order to effect theremoval of this material).

The upper electrode of the resonator component has a lower side which isbound to the piezoelectric layer and an upper side which is exposed.This exposed to surface is then functionalized so that it will react(e.g., bound) with target molecules. In one embodiment, the electricsurface is functionalized with antibody or DNA molecules. This may beaccomplished by forming self-assembling monolayers of various thiols orsulfides on the electrode surface using a chemisorption process. Theantibody or DNA molecules can then be covalently linked to theself-assembled monolayer using an activation process.

Various alternative means for functionalizing the surface of theresonator are also possible. For example, the functionalization of theFBAR device may be achieved by immobilization of biomolecules on anorganic membrane that is pre-coated on the surface of the device, orchemically derivatized such as silylation, esterification, alkylation,or similar processes that are known in the art. The functionalization ofthe FBAR device may also be achieved by direct immobilization ofbiomolecules on a metal or other inorganic film on the surface of thedevice, or self-assembled biomolecular layers such as aminoacid-derivatized fatty acids/lipids on the surface of the device.

Because the biosensor may be used in a “wet” environment, it may benecessary to protect portions of the biosensor other than the exposedresonator surfaces. In other words, the sample which is being tested todetermine the presence of the target biomolecules may be a liquidsample. For some of the components of the biosensor, exposure to aliquid sample may cause problems that could prevent proper operation ofthe biosensor. For example, the liquid sample could cause a shortcircuit between the electrodes of the resonator component, therebypreventing measurement of changes in the frequency response of theresonator. Some embodiments of the invention may therefore also includea protective layer that covers the components of the biosensor, exceptfor the functionalized surface of the resonator component (and possiblythe non-functionalized electrode surface of a corresponding controlbiosensor). This protective layer may be provided by forming a polymermembrane over the components that require protection.

It should be noted that the foregoing description pertains to a single,exemplary embodiment. Alternative embodiments may be formed usingslightly different steps in the described process. A number of thesevariations are included in the description of the foregoing process, andadditional such variations will be apparent to those of skill in the artupon reading the present disclosure. For example, in one alternativeembodiment (FIG. 6), the exposed electrode surface of the resonatorcomponent may be functionalized by coating the surface with anion-selective membrane 145. The ion-selective membrane 145 can then befunctionalized with enzymes such as glucose oxidase. Alternatively, theion-selective membrane can be functionalized with a functional membrane160 that provides transport mechanisms 162 such as carrier molecules,ion pores or channels extracted from biological materials or syntheticbiochemicals.

As noted above, the FBAR biosensor described herein is used by detectingchanges in the frequency response of the resonator that result from theexposure of the biosensor to a sample and subsequent reaction of thefunctionalized surface with the target molecules. In order to determinethe frequency response of the resonator, control circuitry is provided.An exemplary embodiment of a system including an FBAR biosensor andcorresponding control electronics is shown in FIG. 2.

Referring to FIG. 2, a functional block diagram illustrating a biosensorsystem in accordance with one embodiment is shown. In this embodiment,system 200 includes a resonator 210 and control the circuitry 220. Inone embodiment, resonator 210 is as described above. Resonator 210 iscoupled to control circuitry 220 by the electrodes of the resonator. Theelectrodes are coupled to signal generation circuitry 221 and processingcircuitry 222 components of control circuitry 220.

Signal generator circuitry 221 is configured to produce an excitationsignal that is applied to the electrodes of resonator 210. Theexcitation signal has an AC (alternating current) component that causesthe piezoelectric material of the resonator to vibrate. Because of thephysical characteristics of resonator 210, the resonator has acharacteristic frequency response. The frequency response of resonator210 manifests itself in the variability of the electricalcharacteristics of the resonator (e.g., the impedance of the resonator).These electrical characteristics can be measured by processing circuitry222.

The frequency response of resonator 210 has a fundamental resonance at afrequency at which the corresponding wavelength is twice the thicknessof the resonator. The wavelength is equal to the acoustic velocity ofthe piezoelectric material, divided by the frequency. The acousticvelocity of the piezoelectric material depends upon the specificmaterial that is used. For instance, AlN has an acoustic velocity ofabout 10,400 meters per second, while ZnO has an acoustic velocity ofabout 6,330 meters per second. Thus, for a resonator using AlN, if thethickness of the resonator is about 2.5 micrometers, the resonantfrequency is about 2 GHz. If it is desired to adjust the resonantfrequency to a different frequency, this can be achieved by, forexample, changing the piezoelectric material or changing the thicknessof the resonator.

It should be noted that, because the technology used in themanufacturing of FBAR devices is compatible with both Si and GaAs waferprocessing techniques, it is possible to combine these technologies tomake all-in-one biosensors. In other words, it is possible tomanufacture both the resonator and the control circuitry on a singlechip. This may provide additional advantages over the prior art in termsof simplification of the design of the respective components of thebiosensors, improved power efficiency, and so on.

Thus, in one embodiment, biosensor system 200 operates by generating anexcitation signal that includes a plurality of frequencies (notnecessarily at the same time), applying this excitation signal toresonator 210 and then measuring the electrical characteristics of theresonator corresponding to each of the frequencies. For example, signalgenerator circuitry 221 may generate an excitation signal that includesa single frequency which varies as a function of time. In other words,signal generator circuitry 221 scans through a range of frequencies.Processing circuitry 222 may then measure, for example, the impedanceacross resonator 210 as a function of frequency (which is a function oftime). This frequency response (i.e., the impedance of resonator 210 asa function of frequency) may be digitized, stored and compared to abaseline response, or in may be compared to the response of a controlresonator, which would be operated in the same manner.

It should be noted that the excitation signal applied to the resonatormay include various components (e.g., single or mixed frequencies, ortime-variant components). Similarly, the frequency response may bemeasured in terms of various response components (e.g., in-phase andout-of-phase components) or other response characteristics. Suchresponse characteristics may include the steady-state frequency shiftsof the resonators due to changes of mass or electrostatic chargeresulting from the specific binding of the target molecules with theimmobilized biomolecules on the resontaors' surfaces (e.g.,antibody-antigen, DNA hybridization, molecular receptor binding,molecular configurational changes).

The biosensor is useful in the detection of target molecules because, inreacting with the functionalized surface of the resonator, the targetmolecules change the mass and/or electrostatic charge of the resonator,both of which affect the resonance of the resonator. In other words,these characteristics change the vibrational characteristics of theresonator. For example, if the target molecules bind with thefunctionalized surface and thereby effectively increase the mass of theresonator, the resonator will tend to respond less quickly to the forcesgenerated by the applied excitation signal. The resonant frequency willtherefore be lower. Thus, if, prior to the binding of target moleculesto the functionalized surface, a resonator resonates at a frequency f,the additional mass of the target molecules that are bound to thefunctionalized surface will cause the resonator to resonate at afrequency f−Δf. If the frequency response of the resonator is viewed asa function of frequency, this corresponds to a shift of the peakresponse to the left (the lower frequencies).

Referring to FIGS. 3A-3C, a set of diagrams illustrating the binding oftarget molecules to the functionalized surface of a biosensor inaccordance with one embodiment shown. Referring first to FIG. 3A, adiagram illustrating a biosensor prior to exposure to a sample is shown.The biosensor has essentially the same structure shown in FIG. 1,including a support structure formed by substrate 331 and insulatorlayer 332, and a resonator component formed by piezoelectric layer 310sandwiched between electrodes 321 and 322. Electrode 322 of theresonator component is functionalized by antibodies 340, which can bebound to the electrode, for example, by a self-assembling monolayer of athiol. A protective polymer layer 350 (not shown in FIG. 1) covers theresonator component and support structure, except for the functionalizedsurface of the resonator component.

Referring next to FIG. 3B, a diagram illustrating the biosensor of FIG.3A during exposure to a sample is shown. As depicted in this figure,sample 360 contains a variety of different biomolecules, includingantigen molecules (target molecules, represented in the figure bytriangles) and various other molecules (non-target molecules,represented in the figure by circles and squares). The biomolecules aredistributed throughout sample 360, so that some of the biomolecules comeinto contact with the functionalized surface of the biosensor. As thebiomolecules come into contact with the functionalized surface, they mayor may not become bound to the functionalized surface. Morespecifically, if a biomolecule that comes into contact with thefunctionalized surface is an antigen corresponding to the antibodies ofthe functionalized surface, it will be bound to one of the antibodies.If the biomolecule is not the specific antigen corresponding to theantibodies, it will not be bound to the antibodies of the functionalizedsurface.

Referring next to FIG. 3C, a diagram illustrating the biosensor of FIGS.3A and 3B after the sample is removed is shown. It can be seen from thisfigure that, when the sample (e.g., a biological fluid) is removed fromthe biosensor, the non-antigen biomolecules contained in the sample arealso removed. The antigen biomolecules bound to the antibodies remain.These antigens affect the mass and/or electrostatic charge of theresonator component and will therefore change the frequency response ofthe resonator component. Thus, the presence of the antigen biomoleculesis detected by determining whether the frequency response of theresonator component has changed. This is accomplished as describedabove.

It should be noted that, in some cases, removal of the sample from thebiosensor may not ensure that all of the non-target biomolecules havebeen removed from the functionalized surface of the resonator component.These non-target molecules may affect the frequency response of theresonator component and consequently affect the determination of whethertarget molecules were present in the sample. In one embodiment, theeffect of these non-target molecules is compensated for through the useof a control biosensor in addition to the test biosensor. The controlbiosensor is essentially identical to the test biosensor, except thatthe surface of the resonator component is not functionalized to reactwith the target biomolecules. When the sample is tested, both the testbiosensor and control biosensor are exposed to the sample. Anynon-target molecules that are not removed from the test biosensor shouldlikewise not be removed from the control biosensor. Because the controlbiosensor does not bind the target biomolecules, any change in thefrequency response of the resonator component of the control biosensorshould be due to the presence of these non-target biomolecules. Sincethe effect of the non-target biomolecules is known from the frequencyresponse of the control biosensor, this effect can effectively be“subtracted out” of the changes in the frequency response of the testbiosensor.

It should be noted that, while the foregoing example describes the useof two biosensors (a test biosensor and a control biosensor), the twobiosensors may be considered either separate units, or parts of the samebiosensor. The two biosensors may each have their own control circuitry,or they may share all or part of the control circuitry. In the latterinstance, the example may be more easily understood if the term“biosensor” is replaced with the term “resonator.” Both instances arewithin the scope of the invention.

Referring to FIG. 4, a flow diagram illustrating a method for detectingthe presence of target molecules in a sample in accordance with oneembodiment is shown. In this embodiment, a biosensor having a singleresonator is used. The resonator has a functionalized surface that willreact with target molecules in a sample. The method of this embodimentincludes the steps of providing a resonator having a functionalizedsurface (block 410), determining the frequency response of the resonatorprior to exposure to a sample (block 420), exposing the resonator to thesample (block 430), determining the frequency response of the resonatorfollowing exposure to the sample (block 440), comparing the frequencyresponses of the resonator prior to and following exposure to the sample(block 450), and determining the presence of target molecules based uponchanges in the frequency response of the resonator resulting fromexposure to the sample (block 460).

A single resonator is employed in this method, and it is necessary toprovide a baseline frequency response from which changes in thefrequency response can be determined. This baseline is provided in oneembodiment by determining the frequency response of the resonator priorto exposure to the sample. In an alternative embodiment, the baselinemay be provided by testing a plurality of resonators that areidentically manufactured and establishing a composite frequencyresponse, or an average frequency response for resonators having anidentical design. In such embodiment, the composite or average frequencyresponse can be stored in a memory coupled to the processing circuitryso that it can be retrieved and compared to the measured frequencyresponse of the resonator after exposure to the sample. Various othermeans for providing the baseline are also possible.

Referring to FIG. 5, a flow diagram illustrating a method for detectingthe presence of target molecules in a sample in accordance with analternative embodiment is shown. In this embodiment, a biosensor havinga pair of resonators is used. One of the resonators functions as a testresonator, while the other functions as a control resonator. Asdescribed above, the test resonator has a functionalized surface, whilethe control resonator does not. The signal generation circuitry andprocessing circuitry for determining the frequency response of each ofthese resonators may be common. That is, a single set of signalgeneration and processing circuitry can be used in conjunction with bothof the resonators. Alternatively, each resonator may have separatesignal generation and processing circuitry.

This alternative method includes the steps of providing a biosensorhaving dual (test and control) resonators (block 510), exposing theresonator to the sample (block 520), determining the frequency responseof the test resonator and the control resonator following exposure tothe sample (block 530), comparing the frequency responses of the testresonator and control resonator (block 54), and determining the presenceof target molecules based upon differences in the frequency responses ofthe test resonator and control resonator (block 550.

While this alternative method determines the presence of targetmolecules based upon differences between the frequency response of atest resonator and the frequency response of a control resonator, it mayalso be useful to be able to compare either or both of these frequencyresponses to a baseline response. For instance, even though theresonators may be identically designed (except for non-functionalizedsurface of the control resonator), the change in frequency response dueto the presence of target molecules may not be linear. Therefore, it maybe helpful to know not only the magnitude of the difference between thetest and control frequency responses, but also the magnitude of thefrequency response changes resulting from non target molecules (i.e.,the difference between a baseline, pre-sample-exposure frequencyresponse and the control frequency response).

The embodiment illustrated in FIG. 7 includes a pair of resonators, atest resonator 211, and a reference resonator 225. The test resonator211 and the reference resonator 225 are essentially as described aboveexcept that test resonator 211 has a functionalized surface while thereference resonator 225 does not. The reference resonator 225 is used asa control against which the test resonator 211 can be compared. Thus,when both the test resonator 211 and the reference resonator 225 areexposed to a sample, any target biomolecules will affect the frequencyresponse of the test resonator 211, but not the reference resonator 225.Any non-target molecules will equally affect both the test resonator 211and the reference resonator 225 and the corresponding frequencyresponses, so a comparison of the test resonator 211 and the referenceresonator 225 will effectively cancel out any effects resulting fromnon-target molecules.

As noted above, the various embodiments of the present invention mayprovide a number of advantages over the prior art. These advantages mayinclude greater sensitivity and faster response time than other types ofbiosensors, higher resonance frequencies that may provide highersensitivity to changes in mass or in stiffness (resulting from changesin charge), simpler structures than other types of resonators (e.g.,single-crystal quartz microbalance or SAW resonators), better powerhandling characteristics at high frequencies, sharper response peaks(due to reduced parasitic effects, larger surface areas for detection oftarget molecules, and the ability to perform detection of targetmolecules during or after exposure to wet environments. Anotheradvantage of some of the embodiments of the present invention is thatthey may make use of relatively mature technologies (e.g., relating tomethods for designing and manufacturing FBAR devices and integratedcircuits, or functionalizing various surfaces) and may therefore presentfewer difficulties in the design, practice and/or manufacture of therespective embodiments.

The benefits and advantages which may be provided by the presentinvention have been described above with regard to specific embodiments.These benefits and advantages, and any elements or limitations that maycause them to occur or to become more pronounced are not to be construedas critical, required, or essential features of any or all of theclaims. As used herein, the terms “comprises,” “comprising,” or anyother variations thereof, are intended to be interpreted asnon-exclusively including the elements or limitations which follow thoseterms. Accordingly, a system, method, or other embodiment that comprisesa set of elements is not limited to only those elements, and may includeother elements not expressly listed or inherent to the claimedembodiment.

While the present invention has been described with reference toparticular embodiments, it should be understood that the embodiments areillustrative and that the scope of the invention is not limited to theseembodiments. Many variations, modifications, additions and improvementsto the embodiments described above are possible. It is contemplated thatthese variations, modifications, additions and improvements fall withinthe scope of the invention as detailed within the following claims.

1. A system comprising: (a) a film bulk acoustic piezoelectric resonator(FBAR) comprising a layer of piezoelectric material and a pair ofelectrodes coupled to the layer of piezoelectric material, said FBARbeing mounted on an insulator comprising a semiconductive layer andfirst and second, spaced apart insulating layers, said FBAR having afirst edge contacting said first insulating layer and a second edgecontacting said second insulating layer, wherein said semiconductivelayer, said spaced apart insulating layers, and said FBAR define a voidregion therebetween, wherein at least one surface of one of theelectrodes or of the piezoelectric material is functionalized to bindwith target molecules in a liquid sample; and (b) a control circuitcomprising a signal generating circuit to apply an excitation signalthat includes a plurality of frequencies to the pair of electrodes and aprocessing circuit to measure the impedance of the resonator FBAR to thefrequencies of the excitation signal, whereby the presence of thebinding of the target molecule can be detected by observing analteration in the frequency response of the resonator FBAR; (c) a memorycoupled to the processing circuit, the memory having a baseline responsestored therein, wherein the system is configured to detect the targetmolecule comprising a biological molecule.
 2. The system of claim 1,wherein the excitation signal comprises a time variant, single frequencysignal, whereby the plurality of frequencies is applied sequentially. 3.The system of claim 1, wherein the excitation signal comprises a mixedfrequency signal, whereby the plurality of frequencies is appliedsimultaneously.
 4. The system of claim 1, wherein the functionalizedsurface is a surface of the piezoelectric material.
 5. The system ofclaim 1, wherein the functionalized surface is a surface of anelectrode.
 6. The system of claim 1, wherein the substrate comprises asilicon-containing substrate.
 7. The system of claim 1, wherein thefunctionalized surface is functionalized with biologically active orderivatized material.
 8. The system of claim 1, wherein the FBAR iscoupled to the control circuit by the pair of electrodes.
 9. The systemof claim 1, wherein the FBAR has a fundamental resonance at a frequencyat which a corresponding wavelength is twice the thickness of thepiezoelectric layer.
 10. The system of claim 1, wherein the FBAR hasedges and further comprising a support structure wherein the FBAR ispositioned with edges on the support structure.
 11. The system of claim10, further comprising a protective layer that covers the FBAR exceptthe functionalized surface.
 12. The system of claim 1, wherein thefunctionalized surface comprises molecules that bind with targetmolecules in a liquid sample.
 13. The system of claim 1, wherein theexposed surface of the test FBAR electrode is functionalized with an ionselective membrane.
 14. The system of claim 13, wherein theion-selective membrane provides a transport mechanism.
 15. The system ofclaim 1, wherein the electrodes comprise Al, Au, W, Pt or Mo.
 16. Thesystem of claim 1, wherein the piezoelectric material comprises AlN orZnO.